Project Summary/Abstract Neuropeptides are essential modulators of neural circuits governing a wide variety of behaviors including eating, drinking, stress, learning, and reward. While previous genetic and pharmacological studies have provided critical insight into the underlying principles governing peptide function, technical limitations have constrained our ability to effectively probe peptidergic circuits, and many questions remain unanswered. Notably, peptidergic neurons commonly release more than one neuropeptide, and also typically release one fast neurotransmitter, such as glutamate or GABA. Current genetic mouse models and viral tools are unable to effectively and efficiently separate these different neurotransmitter and neuropeptide components. Here I propose to use the neurotensin (NTS) projections from the lateral hypothalamus (LH) to the ventral tegmental area (VTA) as a model peptidergic circuit for the development of novel viral tools that will enable real-time monitoring of peptide release and rapid cell-type specific knockout of peptide-related genes. NTS is known to interact with dopamine neurons in the VTA to promote appetitive behaviors, and we have found that stimulation of NTS projections from the LH to the VTA initiates consumptive and appetitive behavioral phenotypes. These neurons release GABA in addition to NTS, and it is unclear what role each of these transmitter components plays in directing the observed behaviors. We have developed a single-vector conditional viral CRISPR/Cas9 system that enables rapid knockout of any given gene in a cell-type specific manner with high efficiency. I propose to use this system to knock out the gene encoding NTS (Nts) or the gene encoding the vesicular GABA transporter Vgat (Slc32a1) in LH NTS neurons, and will test how removing either component affects the behaviors induced by stimulation of this circuit. I will also use this viral CRISPR method to target NTS receptors in the VTA. In addition, I propose to validate a fluorescent sensor for NTS and use this tool in vivo to monitor peptide release in real time in mice undergoing appetitive behaviors. Completion of this proposal will answer critical biological questions about NTS regulation of behavior via its modulation of the dopamine system and will establish feasibility for generating new viral-based tool kits that can be applied to investigate peptidergic circuits throughout the brain.